The present invention relates to a pharmaceutical composition for use in treatment of a wide variety of malignancies in mammals. The composition of the present invention is non-toxic and targets cancer cells, as opposed to healthy peripheral cells.
Plants may undergo instances of stress, which result in activation of complex genetic pathways that bring about physiological responses appropriate to the stress source. Common stresses to which plants are subjected include extreme UV radiation, osmotic shock, heat shock and pathogen attack. Stress hormones have evolved in plants, which are released in such times of stress and initiate various cascades which end in appropriate responses. Jasmonic acid (JA) and methyl jasmonate (MJ), belong to the group of natural plant stress hormones named xe2x80x9cjasmonatesxe2x80x9d (Sembdner and Parthier, Annu. Rev. Physiol. Plant Mol. Biol., 44, 569-589, 1993). Jasmonic acid is crucial to intracellular signaling in response to injury and methyl jasmonate causes induction of a proteinase inhibitor that accumulates at low concentrations in response to wounding or pathogenic attacks (Farmer and Ryan, Proc. Natl. Acad. Sci., 87, 7713-7716, 1990). Jasmonates have been patented for a variety of uses in plant growth and crop improvement. Application of jasmonates can have a wide range of contradictory effects on virtually all plants. These effects range from inhibition of plant development to promotion of plant processes. U.S. Pat. No. 6,114,284 discloses use of jasmonic acid ester and giberellin to synergistically enhance plant growth and development. U.S. Pat. No. 5,436,226 discloses use of a jasmonate to inhibit sprouting and darkening in tubers after they have been picked, and U.S. Pat. No. 5,118,711 discloses use of methyl jasmonate to repel insects.
Sodium salicylate (SA) is a plant stress hormone of another family, and a central mediator of plant defense responses to pathogens and to injury (Ryals et al., Plant Cell., 8, 1809-1819, 1996).
One response to attack by a microbial pathogen, may be, for instance, programmed cell death termed the hypersensitive response (HR) which results in the formation of a zone of dead cells around the infection site. The layers of dead cells that surround the site of pathogen entry are thought to function as a physical barrier that inhibits further proliferation and spread of the pathogen. A subsequent step of the process involves synthesis of the plant stress hormone sodium salicylate, and accumulation of antimicrobial agents, such as pathogenesis-related proteins and phytoalexins (Dangl et al., Plant Cell, 8, 1793-1807, 1996; Mitler and Lam, Trends. Microbiol., 4, 10-15 1996).
This programmed cell death in response to pathogen attack is reminiscent of programmed cell death known to occur in mammalian cells. Mammalian cells can die by xe2x80x9cunscheduledxe2x80x9d necrosis, which is caused by outside damage and results in cell explosion, or in the more organized manner of apoptosis, also termed xe2x80x9cprogrammed cell deathxe2x80x9d or xe2x80x9ccellular suicidexe2x80x9d (Willingham, J. Histochem. Cytochem., 47, 1101-1109, 1999). In apoptosis, biochemical and morphological events are usually organized in a cascade of very specific and controlled steps, which include fragmentation of the nucleus and shrinkage of the cell, and end with complete splitting of the cell contents to apoptotic bodies (Stewart, J. Cancer Inst., 86, 1286-1295, 1994).
The process of apoptosis is slower then necrosis and happens in a few hours or days, depending on the inducer. This kind of death may be regarded as xe2x80x9ccell suicidexe2x80x9d (Willingham, J. Histochem. Cytochem., 47, 1101-1109, 1999).
Surprisingly enough, the plant stress hormone SA was able to induce intracellular biochemical events typical of a stress response in mammalian cells as well (Schwenger et al., Proc. Natl. Acad. Sci. USA, 4, 2869-2873, 1997; Schwenger et al., J. Cell. Physiol., 179, 109-114, 1999), and was able to induce apoptosis (programmed cell death) in cell lines of human myeloid leukemia, through activation of Caspase-3 (a mammalian cytoplasmic protease essential for the final steps of apoptosis) (Klampfer et al., Blood., 93, 2386-2394, 1999; Willingham, J. Histochem. Cytochem., 47, 1101-1109, 1999; Porter and Janicke, Cell Death Differ., 6, 99-104 1999). SA was likewise able to induce apoptosis in mammalian FS-4 fibroblasts (Schwenger et al., Proc. Natl. Acad. Sci. USA, 4, 2869-2873, 1997), and in human pancreatic cancer (McDade et al., J. Surg. Res., 83, 56-61 1999). The family of drugs of which salicylic acid is a member, non-steroidal anti-inflammatory drugs (NSAID), have potent chemopreventive activity (Morgan, Gut., 38, 646-648 1996; Peleg et al., Dig. Dis. Sci., 41, 1319-1326 1996; Vainio et al., Cancer Epidemiol. Biomarkers Prev., 6, 749-753, 1997).
Many plant genes that respond to environmental and developmental changes are regulated by jasmonic acid, which is derived from linolenic acid by an octadecanoid pathway. Plant defense responses to certain wavelengths of ultraviolet radiation require activation of the octadecanoid defense signaling pathway (Conconi et al., Nature, 383, 826-829, 1996). The release of linolenic acid from the membrane into the cell, and its subsequent conversion to JA, is analogous to signaling pathways in mammalian cells, where releasing of arachidonic acid from the membrane results in synthesis of eicosanoids, such as prostaglandins (Needleman et al., Ann. Rev. Biochem., 55, 69-102 1986). Prostaglandins of the A and J series, which contain a cyclopentanone ring structure, are potent inhibitors of cell proliferation in vitro and are able to suppress tumorigenicity in vivo (D""Onofrio et al., Int. J. Cancer., 51, 481-488, 1992; Gorospe et al., Mol. Cell. Biol., 16, 762-770, 1996). The ability of prostaglandins to arrest growth in a diverse range of tumor cell lines has raised the possibility that they might be useful for treatment of human cancer (Sasaki and Fukushima, Anti-Cancer Drugs, 5, 131-138, 1994). Structural similarity exists between jasmonates and prostaglandins, since both are cyclopentanons, which suggests that JA and MJ may be potent against cancer cells.
The present invention discloses use of members of the plant stress hormone family termed xe2x80x9cjasmonatesxe2x80x9d, for suppressing and killing mammalian cancer cells that represent major types of human malignancies. To the best of the applicant""s knowledge, jasmonates were never studied as anti-cancer agents. Since chemotherapeutic drugs for use in mammalian systems usually work by induction of apoptosis in cancer cells (Bailly et al., Leukemia., 11, 1523-1532, 1997), and jasmonates are thought to be involved in an apoptotic response to plant stress, the applicants tested the ability of jasmonates to suppress replication of mammalian cancer cell lines of clinical importance. The cytotoxicity of jasmonates was compared to that of the plant stress hormone sodium salicylate, which is known to be cytotoxic to mammalian cancer cells.
Chemotherapeutic drugs are often so highly toxic as to leave the patient with numerous side-effects that seriously diminish the patient""s quality of life and impair his function. Chemotherapy regimens can last several months and can be performed repeatedly in cases of relapse, leaving even an ambulatory patient with repeated periods of partial impairment of function. The need exists, therefore, for chemotherapeutic drugs with undiminished potency, yet with a higher degree of specificity towards malignant cells, and fewer side-effects. The present invention discloses use of jasmonate compounds to treat malignancies. Jasmonates are commonly found in minute quantities in many edible plants (Sembdner and Parthier, Annu. Rev. Physiol. Plant Mol. Biol., 44, 569-589 1993), such as tomato, potato, and pumpkin seeds, and thus are non-toxic. Jasmonates are shown by the applicants to be highly specific; inducing apoptosis in clinically important types of cancer cells, yet not effecting the proliferation of normal human cells, such as healthy lymphocytes. Jasmonates are additionally shown by the applicants to be effective in the treatment of lymphoma in a cancer model in mice.
It is the object of the present invention to propose jasmonate compounds as a potent chemotherapeutic drug, with a high degree of specificity towards malignant cells. These and other objects of the present invention will become more apparent from the detailed description of the preferred embodiments, that follows below.
In the present invention, the term xe2x80x9cjasmonatesxe2x80x9d is intended to include the natural plant hormones jasmonic acid and methyl jasmonate, as well as any natural or synthetic derivative and isomers of jasmonic acid and of jasmone. These derivatives have:
1) a lower acyl side chain at C3 (free acid or ester or conjugate)
2) a keto or hydroxy (free hydroxy or ester) moiety at the C6 carbon
3) an n-pentenyl or n-pentyl side chain at C7.
A variety of jasmonates may be used, and include but are not limited to those having the formula: 
wherein n is 0,1, or 2;
R1 is OH, alkoxy, O-glucosyl, or imino,
R2 is OH, O, alkoxy or O-glucosyl, R3, R4 and R5 are H, OH, alkoxy or O-glucosyl, and/or wherein R1 and R2, or R1 and R4 together form a lactone, and further wherein the bonds between C3:C7, C4:C5, and C9:C10 may be double or single bonds.
The present invention relates to a pharmaceutical composition useful for the treatment of cancer in mammals, comprising as an active ingredient a therapeutically effective amount of a jasmonate compound of the formula I: 
wherein:
n is 0,1, or 2;
R1 is OH, alkoxy, O-glucosyl, or imino,
R2 is OH, O, alkoxy or O-glucosyl,
R3, R4 and R5 are H, OH, alkoxy or O-glucosyl,
and/or wherein R1 and R2, or R1 and R4 together form a lactone, and further wherein the bonds between C3:C7, C4:C5, and C9:C10 may be double or single bonds; or a derivative of said formula, wherein the derivative has at least one of the following:
a lower acyl side chain at C3 (free acid or ester or conjugate), a keto or hydroxy (free hydroxy or ester) moiety at the C6 carbon, or an n-pentenyl or n-pentyl side chain at C7.
According to a preferred embodiment of the present invention, the jasmonate is selected from methyl jasmonate, jasmonic acid, jasmone, 7-iso-jasmonic acid, 9,10-dihydrojasmonic acid, 2,3-didehydrojasmonic acid, 3,4-didehydrojasmonic acid, 3,7-didehydrojasmonic acid, 4,5-didehydrojasmonic acid, 4,5-didehydro-7-iso-jasmonic acid, cucurbic acid, 6-epi-cucurbic acid, 6-epi-cucurbic-acid-lactone, 12-hydroxy-jasmonic acid, 12-hydroxy-jasmonic-acid-lactone, 11-hydroxy-jasmonic acid, 8-hydroxy-jasmonic acid, homo-jasmonic acid, dihomo-jasmonic acid, 11-hydroxy-dihomo-jasmonic acid, 8-hydroxy-dihomo-jasmonic acid, tuberonic acid, tuberonic acid-O-xcex2-glucopyranoside, cucurbic acid-O-xcex2-glucopyranoside, 5,6-didehydrojasmonic acid, 6,7-didehydrojasmonic acid, 7,8-didehydrojasmonic acid, cis-jasmone, methyldihydroisojasmonate, dihydro-jasmone, amino acid conjugates of jasmonic acid, and the lower alkyl esters, the carrier ligand conjugates and the sterioisomers thereof.
Further, according to a preferred embodiment of the present invention, the cancer to be treated is selected from prostate cancer, breast cancer, skin cancer, colon cancer, lung cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, kidney cancer, ovarian cancer, bone cancer, liver cancer or thyroid cancer.
Moreover, in accordance with a preferred embodiment of the present invention, the active ingredient is dissolved in any acceptable lipid carrier.
Still further, in accordance with a preferred embodiment of the present invention, the composition additionally comprises at least one other chemotherapeutic agent.
Additionally in accordance with a preferred embodiment of the present invention the composition is prepared for oral administration. In such embodiments, the composition is in a form selected from an emulsion, a solution, a capsule, a tablet.
In another embodiment of the present invention, the composition is prepared for administration by injection. The composition is prepared so as to be suitable for injection intra-muscularly, intra-peritoneally, or intraveneously.
Still further, in certain embodiments, the composition is prepared for topical administration. According to these embodiments, the composition is in a form selected from an ointment, a gel, or a cream.
Moreover, in some embodiments of the present invention, the composition is prepared for administration by inhalation. In other embodiments, the composition is prepared for administration via a suppository.
The present invention further provides a method for treatment of cancer in mammals, comprised of administering a pharmaceutical composition containing as the active ingredient a therapeutically effective amount of a jasmonate compound of Formula I: 
wherein:
n is 0,1, or 2;
R1 is OH, alkoxy, O-glucosyl, or imino,
R2 is OH, O, alkoxy or O-glucosyl,
R3, R4 and R5 are H, OH, alkoxy or O-glucosyl,
and/or wherein R1 and R2, or R1 and R4 together form a lactone, and further wherein the bonds between C3:C7, C4:C5, and C9:C10 may be double or single bonds; or a derivative of said formula, wherein the derivative has at least one of the following:
a lower acyl side chain at C3 (free acid or ester or conjugate), a keto or hydroxy (free hydroxy or ester) moiety at the C6 carbon, or an n-pentenyl or n-pentyl side chain at C7.